The nephrotic syndrome affects both children and adults and may lead to permanent loss of renal function and death. Anatomic abnormalities of the podocyte associated with nephrotic syndrome have been identified for more than 30 years, but the molecular mechanisms by which glomerular epithelial cell pathology causes proteins to leak through the filtration barrier are still poorly understood. Polyanionic molecules are important for the charge selectivity of glomerular filtration. Kerjaschki, Farquhar and colleagues have provided critical information showing that podocalyxin is the major polyanionic molecule of the glomerular epithelial cell surface, and that it plays a key role in the maintenance of foot process structure and function. In spite of its importance, the detailed molecular structure of podocalyxin is not known. The goal of this application is to characterize the molecular structure, function and regulation of expression of podocalyxin. In preliminary studies, podocalyxin monoclonal antibodies have been used to clone overlapping cDNAs from a rabbit renal cortical cDNA library. The full length cDNA of rabbit podocalyxin will be cloned and used to predict the proteins' structure and function. The relationship of the cDNA sequence to the authentic podocalyxin protein will be confirmed by partially sequencing purified podocalyxin protein and comparing the sequence to the nucleotide sequence. Preliminary data indicates heterogeneity of podocalyxin protein in different cell types and tissues. The possible causes of this heterogeneity at the nucleotide level will be explored by looking for multiple genes and alternative splicing. The heterogeneity of podocalyxin protein structure in different cell types and tissues will be determined by Western blot analysis, measurement of electrophoretic mobility and isoelectric point, and by determining the effects of enzymatic desialylation and deglycosylation on gel mobility and binding of anti-podocalyxin monoclonal antibodies. We anticipate that an understanding of podocalyxin structure and regulation at the molecular level will provide new insight into the regulation of glomerular epithelial cell structure, and function and may facilitate development of new treatment strategies for the Nephrotic Syndrome.